Data Sheet

Data Sheet
ALK [5A4]
Concentrated and Prediluted Monoclonal Antibody
Control Number: 901-3041-033115
Catalog Number:
ACI 3041 A, B
API 3041 AA
OAI 3041 T60
0.1, 0.5 ml, concentrated
6.0 ml, prediluted
60 tests, prediluted
Renoir Red
Intended Use:
For In Vitro Diagnostic Use
ALK [5A4] is a mouse monoclonal antibody that is intended for laboratory use in the
qualitative identification of aa 419-520 of ALK protein or ALK-NPM chimeric protein
by immunohistochemistry (IHC) in formalin-fixed paraffin-embedded (FFPE) human
tissues. The clinical interpretation of any staining or its absence should be
complemented by morphological studies using proper controls and should be evaluated
within the context of the patient’s clinical history and other diagnostic tests by a
qualified pathologist.
Summary and Explanation:
ALK (p80) recognizes the formalin-resistant epitope of native anaplastic lymphoma
kinase (ALK) protein. ALK recognizes a 200 kDa transmembrane molecule expressed
only in neural tissues. The ALK reacts with normal ALK protein, as well as with the
chimeric protein ALK-NPM. ALK specifically labels t(2;5)-positive cells giving strong
cytoplasmic staining that is also associated with nuclear staining. Anaplastic large cell
lymphoma (ALCL) is a heterogeneous group of diseases by morphology,
immunophenotyping and clinical presentation. It can be difficult to diagnose because of
its similarity to Hodgkin's lymphoma. However, treatment and prognosis of Hodgkin's
and ALCL is very different, and it is imperative to diagnose correctly. Research has
also shown that a subset of lung adenocarcinomas harbor rearrangements of the ALK
gene that results in the pathologic expression of a fusion protein, most commonly
EMLA-ALK. Patients with ALK-rearranged lung adenocarcinomas are unresponsive to
tyrosine kinase inhibitors that target EGFR, but have shown dramatic improvement in
response to tyrosine kinase inhibitors that target ALK in ongoing clinical trials. The
results from studies comparing FISH, CISH and IHC were concordant. The sensitivity
and specificity of IHC was reported as 100% and 95% respectively. Based on these
findings, the IHC assay using the 5A4 antibody reliably detected non-small cell lung
cancer with ALK rearrangement and may be useful as a screening method to identify
these tumors. Research has shown that ALK stains the majority of CD30+ ALCL. It
has not been shown to stain Hodgkin's disease (Reed-Sternberg cells). ALK should be
used in a panel with CD15, CD30, TIA-1 and EMA.
Principle of Procedure:
Antigen detection in tissues and cells is a multi-step immunohistochemical process.
The initial step binds the primary antibody to its specific epitope. A secondary antibody
may be applied to bind the primary antibody, followed by an enzyme labeled polymer;
or an enzyme labeled polymer may be applied directly to bind the primary antibody.
The detection of the bound primary antibody is evidenced by an enzyme-mediated
colorimetric reaction.
Source: Mouse monoclonal
Species Reactivity: Human; others not tested.
Clone: 5A4
Isotype: IgG1
Total Protein Concentration: ~10 mg/ml. Call for lot specific Ig concentration.
Epitope/Antigen: aa 419-520 of NPM-ALK transcript
Cellular Localization: Cytoplasmic and nuclear staining (dot-like)
Positive Control: Anaplastic large cell lymphoma
Known Applications:
Immunohistochemistry (formalin-fixed paraffin-embedded tissues)
Supplied As: Buffer with protein carrier and preservative
Storage and Stability:
Store at 2ºC to 8ºC. Do not use after expiration date printed on vial. If reagents are
stored under conditions other than those specified in the package insert, they must be
verified by the user. Diluted reagents should be used promptly; any remaining reagent
should be stored at 2ºC to 8ºC.
Protocol Recommendations (manual use):
Peroxide Block: Block for 5 minutes with Biocare's Peroxidazed 1.
Pretreatment Solution: Reveal
Pretreatment Protocol:
Heat Retrieval Method: Retrieve sections under pressure using Biocare’s Decloaking
Chamber, followed by a wash in distilled water; alternatively, steam tissue sections for
45-60 minutes. Allow solution to cool for 10 minutes then wash in distilled water.
Protein Block (Optional): Incubate for 5-10 minutes at RT with Biocare's Background
Primary Antibody: Incubate for 30-60 minutes at RT
Probe: Incubate for 10 minutes at RT with a secondary probe.
Polymer: Incubate for 10 minutes at RT with a tertiary polymer.
Chromogen: Incubate for 5 minutes at RT with Biocare's DAB - OR - Incubate for 5-7
minutes at RT with Biocare's Warp Red.
Counterstain with hematoxylin. Rinse with deionized water. Apply Tacha's Bluing
Solution for 1 minute. Rinse with deionized water.
Protocol Recommendations (ONCORE Automated Slide Staining System):
OAI3041 is intended for use with the ONCORE Automated Slide Staining System.
Refer to the ONCORE Automated Slide Staining System User Manual for specific
instructions on its use. Protocol parameters in the ONCORE Automated Slide Stainer
Protocol Editor should be programmed as follows:
Protocol Name: ALK
Protocol Template (Description): Ms HRP Template 1
Dewaxing (DS Option): DS2
Antigen Retrieval (AR Option): AR1, high pH; 103°C
Reagent Name, Time, Temp.: ALK, 30 min., 25°C
Technical Note:
This antibody has been optimized for use with Biocare's MACH 4 Universal HRPPolymer Detection and ONCORE HRP Detection. Other Biocare polymer detection
kits may be used; however, users must validate incubation times and protocols for their
specific application. Use TBS buffer for washing steps.
The optimum antibody dilution and protocols for a specific application can vary. These
include, but are not limited to fixation, heat-retrieval method, incubation times, tissue
section thickness and detection kit used. Due to the superior sensitivity of these unique
reagents, the recommended incubation times and titers listed are not applicable to other
detection systems, as results may vary. The data sheet recommendations and protocols
are based on exclusive use of Biocare products. Ultimately, it is the responsibility of
the investigator to determine optimal conditions. The clinical interpretation of any
positive or negative staining should be evaluated within the context of clinical
presentation, morphology and other histopathological criteria by a qualified
pathologist. The clinical interpretation of any positive or negative staining should be
complemented by morphological studies using proper positive and negative internal
and external controls as well as other diagnostic tests.
Quality Control:
Refer to CLSI Quality Standards for Design and Implementation of
Immunohistochemistry Assays; Approved Guideline-Second edition (I/LA28-A2)
CLSI Wayne, PA USA ( 2011
Page 1 of 2
ALK [5A4]
Concentrated and Prediluted Monoclonal Antibody
Control Number: 901-3041-033115
1. This antibody contains less than 0.1% sodium azide. Concentrations less than 0.1%
are not reportable hazardous materials according to U.S. 29 CFR 1910.1200, OSHA
Hazard communication and EC Directive 91/155/EC. Sodium azide (NaN3) used as a
preservative is toxic if ingested. Sodium azide may react with lead and copper
plumbing to form highly explosive metal azides. Upon disposal, flush with large
volumes of water to prevent azide build-up in plumbing. (Center for Disease Control,
1976, National Institute of Occupational Safety and Health, 1976) (6)
2. Specimens, before and after fixation, and all materials exposed to them should be
handled as if capable of transmitting infection and disposed of with proper precautions.
Never pipette reagents by mouth and avoid contacting the skin and mucous membranes
with reagents and specimens. If reagents or specimens come into contact with sensitive
areas, wash with copious amounts of water. (7)
3. Microbial contamination of reagents may result in an increase in nonspecific
4. Incubation times or temperatures other than those specified may give erroneous
results. The user must validate any such change.
5. Do not use reagent after the expiration date printed on the vial.
6. The SDS is available upon request and is located at
Follow the antibody specific protocol recommendations according to data sheet
If atypical results occur, contact Biocare's Technical Support at
1. Falini B, et al. ALK expression defines a distinct group of T/Null lymphomas
(“ALK lymphomas”) with a wide morphological spectrum. Am J Pathol. 1998 Sep;
2. Mino-Kenudson M, et al. A novel highly sensitive antibody allows for the routine
immunohistochemistry. Clin Cancer Res. 2010 Mar 1; 16(5):1561-71.
3. Paik JH, et al. Screening of anaplastic lymphoma kinase rearrangement by
immunohistochemistry in non-small cell lung cancer: correlation with fluorescence in
situ hybridization. J Thorac Oncol. 2011 Mar; 6(3):466-72.
4. Kim H, et al. Detection of ALK gene rearrangement in non-small cell lung cancer: a
comparison of fluorescence in situ hybridization and chromogenic in situ hybridization
with correlation of ALK protein expression. J Thorac Oncol. 2011 Aug; 6(8):1359-66.
5. McLeer-Florin A, et al. Dual IHC and FISH testing for ALK gene rearrangement in
lung adenocarcinomas in a routine practice: a French study. J Thorac Oncol. 2012 Feb;
6. Center for Disease Control Manual. Guide: Safety Management, NO. CDC-22,
Atlanta, GA. April 30, 1976 "Decontamination of Laboratory Sink Drains to Remove
Azide Salts."
7. Clinical and Laboratory Standards Institute (CLSI). Protection of Laboratory
Workers from Occupationally Acquired Infections; Approved Guideline-Fourth Edition
CLSI document M29-A4 Wayne, PA 2014.
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